Regulation of differentiation of MEG01 to megakaryocytes and platelet-like particles by Valproic acid through Notch3 mediated actin polymerization

Abstract

Valproic acid (VPA) is one of the HDAC inhibitors used for the treatment of neurological disorders and hematological malignancies. Its role in self-renewal and proliferation of hematopoietic stem cells (HSCs) is well studied, but little is known about its involvement in regulating megakaryopoiesis and thrombopoiesis. In this study, we evaluated the role of VPA in megakaryopoiesis by using MEG-01, a megakaryoblast cell line. Our results show that VPA treatment differentiates MEG-01 cells to megakaryocytes (MK) and platelet-like particles. It was confirmed by augmented expression of MK and PLT-specific markers, higher ploidy, and PLT functionality. We assessed the molecular events underlying megakaryopoiesis. In the present study, we found an upregulation of Notch3 and its downstream target PDGFR-β upon VPA treatment. The direct role of Notch3 in megakaryopoiesis has not yet been studied. PDGFR-β is known to control actin organization during vascular smooth muscle cell differentiation. The actin cytoskeleton plays important role during proplatelet and PLT formation. We found an upregulation of Rac/Cdc42 GTPase and its downstream effectors that are the key players during actin polymerization events. We speculate that VPA induces PLT formation through Notch-3 signaling that in turn modulates actin polymerization that is one of the crucial steps necessary for thrombopoiesis. These studies were also confirmed with knockdown of Notch3 in MEG01 by using ShRNA approach as well as with apheresis-derived CD34⁺ cells. Altogether, these findings provide an evidence for a novel role of Notch3 in regulating platelet formation.

Keywords:

• Megakaryocytes
• notch and differentiation
• platelets
• valproic acid

Acknowledgments

The authors would like to thank Dr. S.C.Mande, Director, NCCS, for support, Mrs. Nikhat Khan for technical help, University Grants Commission (UGC), Government of India, New Delhi, for doctoral fellowship of AD and NCCS core facilities — Flow cytometry, Immunofluorescence Microscopy Facility and experimental animal facility, Central Instrumentation Facility at Savitribai Phule Pune University for electron microscopy, Proteomics facility at National Chemical Laboratory, and Dr. Sameer Melinkeri (Oncologist) from Deenanath Mangeshkar hospital for peripheral blood CD34+ samples. We thank the reviewers for critical evaluation of the manuscript.

Conflict of Interest

The authors declare no competing financial interests.

Supplementary data

Supplementary data for this article can be accessed here.